An Experimental Study On The Preparation Of Pneumococcal Conjugate Vaccine Against Otitis Media Using Pneumolysin As A Protein Carrier And The Immunogenicity Of This Conjugate Vaccine In Infant Mice

Currently, acute otitis media (AOM) is one of the most common infections in childhood and a frequent reason for prescribing antibacterials in infancy. The increasing of drug resistant bacteria against antibiotics is a big problem particularly in China. However, the increase in prevalence of respiratory antibiotics-resistant bacterial pathogens has not been matched by the development of new antibacterial agents. It will result in a vicious circle between overuse of antibiotics and resistance of bacteria. The cost for the treatment of AOM has become a heavy burden in many families. Multiple recurrence of AOM usually leads to chronic otitis media (COM). The recent emergence of drug-resistant strains has provided a strong incentive for preventing AOM by vaccination. Many studies showed Streptococcus pneumoniae was the most common AOM pathogen. At present, the protein-polysaccharide conjugate pneumococcal vaccine has been developed in China. However, tetanus toxoid (TT) or diphtheria toxoid (DT) was regular used as protein carrier in general. Because of some disadvantages of TT and DT as carriers of conjugate vaccine against AOM, pneumolysin has become a promising candidate as a carrier for conjugate vaccine. This study is to research and develop the conjugate vaccine against AOM using pneumolysin carrier.Part 1. The preparation of pneumococcal conjugate vaccine bycoupling 19F polysaccride with BSA or CEAObjectives To explore the technique of preparing the pneumococcal conjugate vaccine and evaluate its immunogenicity in infant mice, and to prepare the coating antigen for evaluating the immunogenicity of PS-Ply conjugate vaccine by ELISA in the future. Methods Type 19F polysaccharide (19F PS) was covalently coupled directly to protein carrier bovine serum albumin (BSA) or chicken egg albumin (CEA) by reductive amination method. The conjugates was purified by sepharose CL-4B chromatography. Al₂(OH)₃ was used as adjuvant to prepare the conjugate vaccine. The 19F-CEA conjugate vaccine was injected into infant mice i.p. with various PS dose, 0.25μg, 1.0μg, 4.0μ.g, per mouse every time, respectively. Every mouse was vaccinated 3 times at 1-week intervals. 19F PS vaccine was injected into the animals as a PS control (4.0μg every time). The negative controls were injected with 0.15M PBS. The antibody titers to 19F PS in mice serum were measured by ELISA using PS-BSA as antigen coating on the plate.Results The peak of A₂₈₀ reading of the conjugate 19F-CEA or 19F-BSA eluate shifted remarkably forward compared with that of 19F PS or the proteins, respectively. The antibody titers gradually increased with the increasment of the PS dose. The control group vaccinated with 19F PS vaccine had almost not antibody response against 19F PS compared with the non-treated group (P>0.05). In contrast, the groups immunized with conjugate vaccine had statistically significant response (P<0.001). In the three study groups, the anti-PS IgG titer of the group with dose 1.0μg was significantly higher than that with dose 0.25μg, whereas there was no significant difference between 1.0μg group and 4.0μg group.Conclusion The procedures to prepare pneumococcal conjugate vaccine have been successfully developed. The conjugate vaccine has effective immunogenicity in infant mice rather than PS vaccine. The titers of anti-PS IgG will increase with the increasement of PS dose of the conjugate vaccine at an extent.Part 2. Preparation of recombinant pneumolysin by genetic engineering technologyObjective To prepare pneumolysin as a new protein carrier of pneumococcalconjugate vaccine.Methods According to the reported gene sequence of Pneumolysin (Ply) by Walker in 1987, a pair of primers which included two restriction enzyme sites were designed. Genomic DNA was isolated from Streptococcus pneumoniae. The gene encoding for pneumolysin was amplified from pneumococcal DNA with PCR. The restriction enzyme digested fragment was linked into the cloning vector PET-28a. The recombinant plasmid DNA containing pneumolysin gene was then transformed into host cell E. coli JM109 (DE3) to express pneumolysin. The positive clones should be seeked out and digested by enzyme for identifying and sequence analyzing. The recombinant pneumolysin(rPly) induced by IPTG was expressed in vivo and purified by using Ni-NTA gel-filtration chromatography . The SDS-PAGE was used to identify the rPly.Results The DNA fragment was amplified from pneumococal chromosomal DNA by PCR and then subcloned into the expression vector pET-28a. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and agarose gel electrophoresis. The pneumolysin protein was successfully expressed by JM109(DE3) with IPTG inducement. The relative molecular mass of the expressed product was 52kDa. The purity grade of rPly purified by Ni-NTA gel-filtration chromatography was above 90%.Conclusion The rPly could be obtained by genetic engineering technology. The purity and the yield of rPly purified by Ni-NTA gel-filtration chromatograph were good enough for the conjugation with PS.Part 3. The preparation of pneumococcal conjugate vaccine using pneumolysin as a protein carrier and the immunogenicity ofthe vaccine in infant miceObjectives To prepare pneumococcal conjugate vaccine using pneumolysin as a protein carrier and evaluate the immunogenicity of the conjugate vaccine in infant mice.Methods The pneumolysin obtained via genetic engineering technology was detoxified by 0.2% or 0.5% formalin respectively. Pneumococcal polysaccharide (PS,19F serotype) was conjugated with detoxified Ply using amino reductive method. The conjugate was purified via SepharoseCL-4B chromatography. Then the adjuvant Al₂(OH)₃ was mixed with the conjugate before vaccination of the mice. The study groups of infant mice of 3 weeks old were vaccinated with the conjugate vaccine 1 to 3 times at 1-week intervals. The infant mice injected with 0.15M PBS i.p. served as negative control group. The antibody response to 19F PS or Ply was measured by ELISA respectively.Results Pneumolysin could be detoxified by 0.2% or 0.5% formalin. The peak of A₂₈₀ reading of the conjugate 19F-Ply eluate shifted remarkably forward compared with that of 19F PS or the protein Ply. So the PS(19F serotype) was successfully conjugated with Ply. The GMTs of IgG against 19F PS of three groups which were vaccinated with conjugate vaccine for 1 to 3 times were 32.25, 42.07, 72.41, respectively, and it was only 8.9 in the control group. The GMTs of IgG against Ply were 36.21, 80.71 and 138.3, and it was 22.37 in the control group. There was a significant difference of antibody titer against 19F PS or Ply between vaccinated and non-vaccinated mice (P<0.01), despite of the times of vaccination. Conclusion The 19F PS-Ply conjugate vaccine produced using amino reductive method can induce specific immune response to both 19F PS and Ply in infant mice. It can be considered as preferable vaccine candidate against otitis media for otitis prone children.